Abstract The nasopharyngeal tract is a major portal entry of debilitating and potentially lethal pathogens including Bacillus anthracis and influenza virus. Mucosal vaccines capable of promoting antibody and cytotoxic T cell responses in mucosal tissues, in addition to the general bloodstream, protect more effectively against respiratory pathogens than classical injected vaccines. Therefore, there is a need for safe mucosal adjuvants and vaccine delivery systems. Previous work on this grant focused at defining murine nasal-associated lymphoreticular tissues (NALT) as inductive sites for immune responses targeting the nasopharyngeal tract. We used cholera toxin and developed novel derivatives lacking ADP ribosyl transferase activity to circumvent the reactogenicity of this enterotoxin adjuvant. In addition, we have assessed the action of anthrax toxins on murine NALT. These studies have also included the characterization of antibodies (Abs) to anthrax protective antigens with special emphasis on the potential to protect mucosal tissues. A total of five original Specific Aims were successfully addressed. A major and unexpected finding during the course of our studies was the fact the nasal co-administration of Bacillus anthracis protective antigen together with a mutant of the cAMP-inducing Bacillus anthracis edema factor enhanced mucosal and systemic immunity against these two molecules. Furthermore, we found that the edema toxin (EdTx or protective antigen plus edema factor) derivative enhanced mucosal and systemic immunity to co-administered unrelated antigens such as recombinant Yersinia pestis F1-V antigen. Unlike the ganglioside-binding enterotoxin cholera toxin, neither EdTx nor its derivatives target central nervous system tissues after nasal application. In this renewal grant, we will address the overall hypothesis that sublingual application of EdTx derivatives will induce NALT-based immunity and protect against respiratory pathogens, without the adverse effects often associated with nasal application of enterotoxins. Specific aim one will establish the adjuvant activity of edema factor derivatives co-administered with protective antigen (PA) via the sublingual route for enhanced immunity to anthrax toxin components. Specific aim two will characterize protective immunity to respiratory viral infection afforded by sublingual immunization with EdTx derivatives as adjuvant. Studies in specific aim three will identify inductive sites for the generation of secretory IgA (SIgA) Abs and mucosal immunity after sublingual immunization with EdTx-derivatives. Finally, specific aim four will determine molecular signals underlying the induction of SIgA responses by EdTx-derivatives as sublingual adjuvant. This grant will unravel the mechanisms by which EdTx derivatives act as adjuvant for sublingual vaccines and induce NALT-based immunity. We will also validate a new route for mucosal vaccine delivery, as well as new PA-based mucosal adjuvant(s) for the induction of immunity against respiratory pathogens.